Adaptation Displacement: While most of the Tezuka characters who show up here did make earlier stateside debuts in one form or another (even if only as extras in other Astroboy productions), for a time this was what all of them (with the possible exception of Black Jack, whose OAV series enjoyed modest success in the West during the late 90s/early 00s anime boom) were best known for in the English-speaking world, though Vertical Inc.'s recent blitz of high quality Tezuka manga translations has done a lot to change that.
Fridge Brilliance: The Final Boss music is titled "World's Strongest Robot". This both refers to Pluto, as he's the leader of the "World's Strongest Robots"; and Garon, who the Character List refers to as "the most powerful super robot sent to Earth".
Tear Jerker: The death of Dr. Tenma's son, Tobio; And him grieving for his son as he creates Astro in his image. And this is the first scene in the game.
The first ending of the game, where Death Mask arrives and destroys all robots on Earth, including Astro; leaving behind a world completely ravaged by years of war between humans and robots. Don't worry though, it's not over yet...
The story of Daichi and Prime Rose. Which becomes even more so when you learn later that the two of them aren't actually gone.
Drake himself is truly a tragic villain, having been pushed over the edge entirely by the belief that his daughter was kidnapped and killed by a robot, leading to his hatred of robots with him vowing to do everything in his power to avenge her death.
North in Hard Mode, mostly because his first phase basically forces you to waste all three of your Supers.
Atlas, when fighting him with Dr. Black Jack at the bottom of the map, which leaves you with less space to fight him.
The jousting duel with Blue Knight, which requires landing a few hits while being a One-Hit-Point Wonder regardless of your life level. Part of this is due to being a Guide Dang It!, and another part is due to having to replay a lot of the level if you die.
The Untwist: Blue Knight being President Rag. The gratuitous flashback makes it screamingly obvious.